Retroviruses are viruses that contain a single strand of RNA as the genetic material rather than DNA. The single-stranded RNA genome of each of these viruses gives rise to a double-stranded DNA molecule after the virus infects a susceptible host. This DNA replica of the viral genome then introduces itself permanently into a chromosome of the successfully infected cell and replicates in that host chromosome.
The retroviruses discussed hereinafter and in the claims may be further defined as being replication-defective retroviruses. Thus, these viruses do not themselves contain a gene encoding the reverse transcriptase usually required to permit the viral RNA genome to be translated into a DNA that can be introduced into a chromosome of the infected host. Rather, the retroviruses discussed hereinafter typically must be complimented in their infection by a so-called helper virus that is replication-competent. That second virus contains the gene that encodes the reverse transcriptase enzyme that incorporates the genomic materials from both viruses into the successfully infected host cells to transform those cells.
For ease in understanding, the replication-defective retroviruses will be discussed hereinafter and in the claims merely as retroviruses with the understanding that they are replication-defective and require the assistance of a helper virus for successful infection and transformation of host cells. This usage of the term retrovirus is known in the art and has been used in the art as such without further explanation.
Some members of the retrovirus family are highly oncogenic as judged by their ability to cause the formation of solid tumors within a short period of time after being inoculated into the host. These viruses can also cause "cancerous" changes in cells grown and cultured in the laboratory; such changes are called "transformations" and provide a reliable in vitro biological assay for oncogenic viruses. Several such viruses have been isolated from chickens, turkeys, mice, rats, cats and monkeys.
A single gene, the oncogene, located on the genome of these highly oncogenic viruses is responsible for the tumorgenic potential of the virus. In the case of several viruses, the protein products of their oncogenes, referred to herein as oncoproteins, have been immunologically identified by taking advantage of the fact that serum from an animal bearing a virus-induced tumor contains antibodies directed against those oncoproteins.
A rapidly growing body of evidence indicates that the oncogenes of retroviruses are closely related to and are derived from specific genetic loci in the normal cellular genetic information of all vertebrates.
Interest in oncogenes has steadily risen in the last decade. Although RNA tumor viruses have been implicated as the causative agents of experimentally induced neoplasia in chickens for over 50 years, it was not until the mid 1970s that mechanisms of virally induced neoplasia began to emerge [Bishop (1983) Ann. Rev. Biochem. 52:301-54]. According to one such mechanism, replication-competent avian viruses and defective mammalian viruses had captured cellular genes that provided the viruses with a transforming potential.
Molecular hybridization studied using specific nucleic acid probes, followed by genetic cloning of viral oncogenes and their cellular relatives by recombinant DNA technology, have established the kinship between retroviral oncogenes (v-onc) and cellular oncogenes (c-onc) found in all normal vertebrate cells. Molecular analysis of the several retroviruses thus far isolated has revealed more than two dozen different oncogenes. In most cases, a corresponding cellular to the retroviral oncogene or oncoprotein has been isolated.
For example, the human EJ or T24 bladder carcinoma oncogene was identified as the homolog of the transforming gene of Harvey murine sarcoma virus (ras.sup.Ha) and also of the BALB sarcoma virus (bas) [Parada et al., Nature, 297, 474-478 (1982); Der et al., Proc. Natl. Acad. Sci USA, 79, 3627-3634 (1982); and Santos et al., Nature, 298, 343-347 (1982)]. In addition, the oncogene of the human carcinoma cell line LX-1 was found to be homologous to the transforming gene of Kirsten strain of murine sarcoma virus (ras.sup.Ki) [Der et al., above]. Still further, the v-onc for a c-onc designated fps of avian origin is represented at least twice among a limited number of avian retrovirus isolates; its mammalian cognate designated fes in feline species is found in two different strains of feline sarcoma viruses.
The homology [Doolittle et al., (1983) Science 221:275-277; Waterfield et al., (1983) Nature 304:35-391] between the gene product of the sis oncogene and one of the chains of platelet-derived growth factor provided the most solid link between malignant transformation by oncogenes and stimulation of normal cell division by growth factors. This identity between oncogene products and growth factors and cellular receptors was further substantiated with sequence analysis of the epidermal growth factor cellular receptor [Downward et al., (1984) Nature 307, 521-527; Ullrich et al., (1984) Nature 309:418-425] that was found to be the normal homologue of erb B. Furthermore, immunological cross-reactivity of fms antibodies with colony stimulating factor-1 receptor [Sherr et al., (1985) Cell: 665-676] as well as protein kinase homology with the insulin-receptor [Ullrich et al., (1985) Nature:313, 756-7611] and platelet derived growth factor receptor [Yarden et al., (1986) Nature 323; 226-232] indicated the kinase activity of many of the sequenced oncogenes would be important in the signal transduction of several growth factors.
Sequencing of oncogenes captured by retroviruses or identified via transfection experiments greatly extended the number of kinase family members. [Hunter et al., (1985) Ann. Rev. Biochem. 54:897-930.] This sequence analysis suggested the number of kinase-related proteins would be large and the family members could be divided into subgroups based upon sequence homology and overall structural similarities. The kinase family can be conveniently divided into gene products that do or do not have extracellular (hormone/growth factor) binding domains.
The close similarity between the kinase portion of src and yes has been apparent for several years. [Kitamura et al., (1982) Nature 297:205-208.] Recently, sequencing of additional genes has extended this homology to fgr, [Naharro et al., (1984) Science 222;63-66] lck, [Marth et al., (1985) Cell 43:393-404. syn, [Semba et al., (1986) Proc. Natl. Acad. Sci. USA 83:5459-54631 ] and lyn [Yamanashi et al., (1987) Mol. and Cell Biol. 1:237-243]. All six of these genes encode proteins of approximately the same size 55-65 kd, and the genes share intron/exon borders indicating they evolved from the same ancestral protooncogene. However, each gene is located on a separate chromosome and expresses different proteins in different tissues.
Many additional kinase family members can also be placed into subgroups. Mos [Van Beveran et al., (1981) Nature 289:258-262] is closely related to pim-1 [Selten et al., (1986) Cell 46:603-611], one of the preferred integration sites of Moloney leukemia virus. Abl [Reddy et al., (1983) Proc. Natl. Acad. Sci. USA 80:3623-3627] is closely related to arg [Kruh et al., (1986) Science 234:1545-1547]. Fes [Hampe et al., (1982) Cell 30:775-785] and fps [Shibuya et al., (1982) Cell 30:787-795]. represent the mammalian and avian counterparts of the same gene. Similarly, raf [Sutrave et al., (1984) Nature 309:85-88] and mil [Mark et al., (1984) Science 224:285-289] are mammalian and avian homologues of the same gene. They are closely related to A-raf/pks [Huleihel et al., (1986) Mol. and Cell Biol. 6:2655-2662; Mark et al., (1986) Proc. Natl. Acad. Sci. USA 83:6312-6316].
A subgroup that does not have a viral counterpart contains genes that encode protein kinase C, the receptor for phorbal esters. There are at least three closely related genes comprising this subgroup [Coussens et al., (1986) Science 233:859-866; Knopf et al., (1986) Cell 46:491-502]. Moreover, one of the genes can encode two proteins via alternative exon usage [Ohno et al., (1987) Nature 325:161-166]. Other more distantly related cytoplasmic kinases include cAMP- and cGMP-dependent protein kinase [Shoji et al., (1981) Proc. Natl. Acad. Sci USA 78:848-851; Takio et al., (1984) Biochemistry 23:4207-4218], as well as myosin light chain kinase [Takio et al., (1985) Biochemistry 24:6028-6037]. Several transmembrane kinases have also been sequenced in the past few years.
A gene closely related to the human epidermal growth factor receptor (HER) has also been found in humans (HER-2) [Coussens et al. (1985) Science 230:1132-1139] and rats (neu) [Bargmann et al., (1986) Nature 319:226-230]. The growth factor that binds to ros [Neckameyer et al., (1985) J Virol. 53:879-884] is not known although the sequence is most closely related to the insulin receptor (HIR) [Ullrich et al., (1985) Nature:313, 756-761]. The colony stimulating factor 1 receptor, FMS [Hampe et al., (1984) Proc. Natl. Acad. Sci. USA 81:85-89], forms a subgroup with kit [Besmer et al., (1986) Nature 320:415-421] and the receptor for platelet-derived growth factor, PDGF-R [Yarden et al., (1986) Nature 323:226-232]. In addition, sequences for the trk [Martin-Zanca et al., (1986) Nature 319:743-748] and met-8 [Dean et al., (1985) Nature 318:385] oncogenese have been published, although the corresponding growth factors are not known.
A similar although not as extensive expansion has also been seen for the nucleotide binding proteins represented by the ras oncogene family. Sequence data indicate bas [Reddy et al., (1985) J. Virol. 53:984-987] is the mouse form of H-ras [Dhar et al., (1982) Science 217:934-937], and that the H- and K-ras products differ principally at the carboxyl region [Tsuchida et al., (1982) Science 217:937-939]. Through alternative exons K-ras can encode 2 proteins (4A and 4B) [McGrath et al., (1983) Nature 310:501-506]. A third member, N-ras, also diverges from H- and K-ras in this region [Taparowsky et al., (1983) Cell 34:581-586]. Another closely related gene is R-ras [Lowe et al., (1987) Cell 48:137-146], although this gene is closely related to the three ras genes that have evolved from the same ancestral gene, R-ras has different intron/exon boarder. Another gene, rho [Madule et al., (1985) Cell 41:31-40], has scattered regions of homology with ras. Furthermore, a third group, ral, also has similar regions of homology [Chardin et al., (1986) EMBO J. 5:2203-2208]. Moreover, a yeast gene ypt [Gallwitz et al., (1983) Nature 306:704-707] has regions of homology with ras and this gene is distinct from the two yeast genes that have extensive homology with ras; i.e., they are more like R-RAS.
Other genes that also have homology with ras include the G proteins [Itoh et al., (1986) Proc. Natl. Acad. Sci. USA 83:3776-3780] as well as transducin and elongation factor, Tu [Lochrie et al., (1985) Science 228:96-99]. The G proteins are composed of subunits that stimulate (G.sub.s) and inhibit (G.sub.i) adenylate cyclase. Another related protein (G.sub.o), has an unknown function. These proteins exists in a variety of different forms that have closely related sequences.
The nuclear proteins myb [Rushlow et al., (1982) Science 216,1421-1423], myc [Colby et al., (1983) Nature 301:722-725] and fos [van Straaten et al., (1983) Proc. Natl. Acad. Sci. USA 80:3183-3187] comprise another family of oncogenes that are related more by cellular location than sequence. However, additional genes related to these oncogenes have been identified. N-myc [Stanton (1986) Proc. Natl. Acad. Sci. USA 83:1772-1776] and L-myc [Nau et al., (1985) Nature 318:69-73] sequences have been published, and unpublished related sequences have been identified. Moreover, the sequences are distantly related to fos. A related fos (r-fos) [Cochran et al., (1984) Science 226:1080-1082] sequence has been published, and unpublished data indicate a phosphorylase inhibitor has limited homology as does the jun oncogene.
Another group of nuclear oncogene-related proteins include steroid and thyroid hormone receptors. Although only one sequence related to erb A has been published [Sap et al., (1986) Nature 324:635-640; Weinberger et al., (1986) Nature 324:641-646], hybridization studies indicate at least two related sequences are present in the human genome [Weinberger et al., (1986) Nature 324:641-646]. Steroid receptor sequences indicate erb A (the thyroid hormone receptor) is part of a superfamily that includes several receptors (estrogen, glucocorticoid, progesterone, aldosterone) [Greene et al., (1986) Science 231:1150-1153; Hollenberg et al., (1985) Nature 318:635-641; and Connelly et al., (1986) Science 233:767-770].
In the growth factor group only the PDGF-1 chain [Doolittle et al., (1983) Science 221:275-277 and Waterfield et al., (1983) Nature 304:35-39] has sequence homology to sis (PDGF-2). However, other growth factors [Gregory (1975) Nature 257:325-327; Marguardt et al., (1983) Proc. Natl. Acad. Sci. USA 80: 4684-4688] (EGF and TGF) bind to the product of the erb B protooncogene, and CSF-1 [Kawasaki et al., (1985) Science 230:291-296] binds to the fms protooncogene. Moreover, TGF [Derynk et al., (1985) Nature 316:701-705], forms another subgroup by virtue of homologies with Mullerian inhibitory substance [Cate et al., (1986) Cell 45:685-698], and the three chains that are found in the various forms of inhibitin [Mason et al., (1985) Nature 318:659-663 and Vale et al., (1986) Nature 321:776-779].
Finally, sequences representing two of the preferred integration sites of MMTV have been published [Van Ooyen et al., (1984) Cell 39:233-240 and Moore et al., (1986) EMBO J. 5:919-924].
Thus, in the past few years, the number of related published sequences has increased dramatically. These sequences suggest that a limited number of pathways controlling cell division and differentiation exist but that many different members may participate in this control.
An example of transduction of only a portion of a cellular gene by a retrovirus is the erb B oncogene. The erb B oncogene is highly homologous to a portion of the ECG receptor [Ullrich et al., Nature 309:418 (1984)], as already noted. Sequence analysis of the entire receptor gene demonstrates the relatedness of erb B with the entire intracellular domain, the transmembrane domain, and a portion of the extracellular doman.
The protein encoded by the viral oncogene and the corresponding, homologous protein within the host cell are both referred to herein as oncoproteins, although the cellular oncoprotein is typically larger and is present in small quantities in normal cells, and thus need not only be associated with neo-plastic states. In addition, oncoproteins encoded by related oncogenes can have different molecular weights, e.g., the p85 and p108 oncoproteins encoded by v-fes.sup.ST and v-fes.sup.GA, respectively, and the 100-105 kilodalton (also kd or K dalton) protein of normal mink cells thought to be encoded by the c-fes gene. [Sen et al., Proc. Natl Acad. Sci. USA, 80, 1246-1250 (1983).] The term oncoprotein is thus used generally herein for proteins whose genes and amino acid residue sequences are homologous, at least in part, as discussed hereinafter.
The oncoprotein is generally not present in the virus particle that infects the cell, but is only expressed after infection and transformation. The corresponding cellular oncoprotein is expressed at most minimally in normal cells and to a greater extent in neoplastic cells. Thus, the oncoprotein cannot typically be obtained from the virus. In addition, isolation of oncoproteins from cells is made difficult because of small amount present, the complex mixture of proteins found in normal cells, and the relatively small amount of such proteins present even in transformed cells.
Oncoproteins encoded by v-onc and c-onc genes thus typically contain large sequences of amino acid residues that are homologous, but nevertheless are not usually identical. In addition, oncoproteins encoded by genes of different viral strains, each of which contains ostensibly the same oncogene, have been found to have slight variations in their amino acid residue sequences as exemplified above, and by the four published sequences of the ras gene which differ at the position of the twelfth amino acid residue. Thus, even when oncoproteins are in hand, it may be difficult to distinguish among them.
Immunologically induced receptor molecules such as monoclonal and polyclonal antibodies or the idiotype-containing portions of those antibodies are useful in purifying protein ligands to which they bind, as diagnostic reagents for assaying the presence and quantity of the protein ligands, as well as for distinguishing among homologous protein ligands.
The difficulties associated with obtaining quantities of oncoproteins typically militate against the preparation of receptors to those oncoproteins, although whole cell-induced monoclonal antibodies to v-fes and v-fps encoded oncoprotein have been reported by Veronese et al., J. Virol., 43, 896-904 (1982). In addition, even were whole proteins available for use as immunogens for inducing the production of such receptors, the use of large protein molecules as immunogens produces antisera containing polyclonal antibodies to the numerous epitopes of the large protein molecules.
Hybridoma and monoclonal antibody techniques utilizing whole proteins or large protein fragments as immunogens have been useful in narrowing the immunological response to such immunogens. However, such technology as heretofore practiced has been extremely time consuming and has provided only a relatively small number of hybridomas that secrete useful antibodies that recognize the immunogen. Moreover, even when successful, such techniques cannot be predictive of the chemical identity of epitope to which the receptor molecules are raised. Consequently, even after immunogen-recognizing receptors are produced, the obtaining of receptors to specific, chemically identified epitopic portions of the protein ligand has been a hit or miss operation that still further reduces the number of useful hybridomas that are ultimately produced.
Arnheiter et al., Nature, 294, 278-280 (1981) reported on the production of monoclonal antibodies that were raised to a polypeptide that contained 56 amino acid residues and corresponded in amino acid residue sequence to the carboxyterminal portion of an intact interferon molecule. The 56-mer polypeptide thus corresponded to approximately one-third of the sequence of the intact molecule.
Arnheiter et al. reported on the production of eleven monoclonal antibodies. However, only one of those eleven monoclonal antibodies bound both to the polypeptide immunogen and also to the intact interferon molecule. In addition, that binding was not very strong as judged by the 3000-fold excess of intact interferon required to compete the antibody away from the synthetic polypeptide. None of the other monoclonal antibodies bound to the intact molecule.
In addition, the production of the hybridomas secreting those monoclonal antibodies required the spleens from three immunized mice. The low yield of the desired interferon-binding monoclonal antibodies, and the fact that three mouse spleens were needed for the preparation of those hybridoma cell lines indicates that those workers were relatively unsuccessful in their efforts.
Lerner et al. have been successful in obtaining protection of animals by the use of vaccines against pathogens by utilizing synthetic amino acid residue sequences of short to moderate length as immunogens. See Sutcliffe et al., Science, 219, 495-497 (1983).
However, it must be understood that until the present invention, successful preparation of hybridomas and their secreted monoclonal receptors differs from the successful preparation of a vaccine containing oligoclonal receptors. Thus, for a high yield monoclonal antibody preparation, it is necessary to stimulate B-cells to secrete large amounts of avid antibodies. On the other hand, for a synthetic vaccine, a wider spectrum of oligoclonal antibodies may be produced in smaller amounts and with lower avidities. In addition, protection of an animal against a pathogen typically requires both T-cell and B-cell activations so that a cellular response and a humoral response, respectively, can be induced in the animal.
A popular explanation for the success of synthetic polypeptide-containing vaccines in generating antibodies that recognize intact proteins and protect animal hosts involves a stochastic model in which the diversity of the immune response allows the observation of an infrequent event; i.e., the polypeptide adopting the confirmation of its corresponding sequence in the native molecule. The concept that moderate-length polypeptides can frequently conform to native structures is contrary to theoretical and experimental studies. Rather, such polypeptides are thought to exist as an ensemble of a large number of transient conformational states that are in dynamic equilibrium. T-Cell activation by, and B-cell production of antibodies raised to, some of that conformational ensemble have been believed sufficient to provide protection upon vaccination.